Analysis of Phylogenetic Relationships in The Walnut Family Based on Internal Transcribed Spacer Sequences and Secondary Structures(ITS2) Zhongzhong Guo Tarim University Qiang Jin Tarim University Zhenkun Zhao Tarim University Wenjun Yu Tarim University Gen Li Tarim University Yunjiang Cheng Tarim University Cuiyun Wu Tarim University rui Zhang ( [email protected] ) Tarim University https://orcid.org/0000-0002-4360-5179 Research Article Keywords: Base sequence, Evolution, Juglandaceae, Ribosomal spacer, Secondary structure Posted Date: May 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-501634/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/23 Abstract This study aims to investigate the phylogenetic relationships within the Juglandaceae family based on the Internal Transcribed Spacer's primary sequence and secondary structures (ITS2). Comparative analysis of 51 Juglandaceae species was performed across most of the dened seven genera. The results showed that the ITS2 secondary structure's folding pattern was highly conserved and congruent with the eukaryote model. Firstly, Neighbor-joining (N.J.) analysis recognized two subfamilies: Platycaryoideae and Engelhardioideae. The Platycaryoideae included the Platycaryeae (Platycarya+ (Carya+ Annamocarya)) and Juglandeae (Juglans-(Cyclocarya + Pterocarya)). The Engelhardioideae composed the (Engelhardia+Oreomunnea+Alfaroa)). The Rhoiptelea genus was generally regarded as an outgroup when inferring the phylogeny of Juglandaceae. However, it is clustered into the Juglandaceae family and showed a close relationship with the Platycaryoideae subfamily. Secondly, the folded 3-helices and 4-helices secondary structure of ITS2 were founded in the Juglandaceae family. Therefore, these ITS2 structures could be used as formal evidence to analyze Juglandaceae's phylogeny relationship. The morphology based on the secondary structure nicely coincided with previous investigations. This study further conrms that ITS2 can serve as a valid basis for parsing evolutionary relationships in higher woody fruit trees. Introduction Several molecular (nuclear and plastid) studies have been conducted to infer phylogenetic relationships at a low taxonomic level. The coded ribosomal RNA (rDNA) repeat has been useful in intra- and inter- specic phylogenetic studies within the angiosperms. In eukaryotic cells, the highly conserved rRNA genes are abundant and are arranged in a tandem series at the nucleolar organizer region (NORs) of chromosomes. Each unit is comprised of a non-transcribed spacer known as an intergenic spacer (IGS), the transcription unit coding for the precursor of the rRNA, so the composed basic unit contains the following segments is 5'-IGS-18S-ITS1-5.8S-ITS2-26S-3’[1–4]. The two internal transcribed spacers (ITS1 and ITS2), which are part of the nuclear ribosomal cistron, but are not incorporated into mature ribosomes, have been regarded as valuable targets when used as markers in systematic studies. Sequencing of rDNA regions, including the small ribosomal subunit 5.8S and ITS, have been widely applied in phylogenetic classications because of the relatively higher mutation rate [5–8]. Furthermore, the ITS2 sequence has been widely used to infer phylogenetic relationships at low intra- and inter-hierarchical levels even at the species and genus ranks based on the predominance nature of hypervariable subregions, biparental inheritance, and intergenomic variability [3]. However, the typical challenge on the alignment of ITS2 sequences arises from closely related and recently radiated taxa due to the highly conserved nature of some ITS2 subregions and other parts that seem to vary high randomly substitution [9]. When phylogenetic inferences are limited to subregions with precise alignment, effective information content may be lost. To gain more insights into the processes shaping expansion segments, studies aimed at comparing the evolution of ITS2 primary sequences and secondary structures are required [10, 11]. The ITS domain brings the large and small ribosomal submits into proximity for mature Page 2/23 processing; the ITS domain's secondary structures were considered important in dening the cleavage sites for the rRNA release. So, the ITS2 secondary structures did not incorporate into mature ribosomes and were relatively highly conserved [9, 11]. Hence, it is crucial to identify the ITS2 sequence and its secondary structure when performing alignments and reconstruct phylogenies. The Juglandaceae, also known as the walnut family, comprises woody trees and shrubs and belongs to the order Fagales. The Juglandaceae is a vital nut and timber producer in temperate regions of Europe, Asia, South America, and North America. The Juglandaceae consists of 9 genera and 72 species in the world. Based on Manos's results [12], the Juglandaceae family comprises two major subfamilies; the Juglandoideae and the Engelhardioideae. Within the Engelhardioideae subfamily, the genus Engelhardia was paraphyletic to the sub-clade composed of Oreomunnea + Alfaroa genus. In the Juglandoideae, the full Juglandeae clade (Carya-(Juglans-(Cyclocarya + Pterocarya))) was the sister group to the Platycaryeae genus. However, Schaarschmidt [13] suggested that the Juglandaceae comprised the Platycaryoideae and Juglandoideae. The Platycaryoideae contains two sister groups, the Platycaryeae and Engelhardieae, and the Juglandoideae includes the Juglans, Pterocarya, and Cyclocarya genera. The germplasm in the Juglans genus is of great economic value in the Juglandaceae family. Based on leaf and ower morphology by Dode [14], and fruit and nut morphology by Manning's [15], the Juglans genus can be divided into four sections, namely, Dioscaryon Dode (traditional Juglans), Rhysocaryon Dode (black walnuts), Cardiocaryon Dode (Asian butternuts), and Trachycaryon Dode (American butternut). Aradhya [16] suggested that the Juglans section includes the cultivated Persian (English walnut, Juglans regia L.), and iron walnut (Juglans sigillata Dode), which is originated in the Southern Chinese province of Yunnan and Tibet. Based on results published by Ross-Davis [17], the J. cinerea was located close to J. nigra and other members of the Rhysocaryon section than to the Asian members of the Cardiocaryon section. Previous studies based on fossils, morphology, chemical, chromosome, and sequence analysis have established a framework for the Juglandaceae family [12] [18, 19]). The present cladistic analysis included 51 taxa across six genera (including Juglans, Cyclocarya, Pterocarya, Platycarya, Carya, Annamocarya), with the outgroup Rhoiptelea and the partial subfamily Engelhardioideae (Engelhardia, Alfaropsis, Oreomunnea). The present study focused on the features of the secondary structure of ITS2. The objective of the study was to provide new information for the phylogenetic status of the Juglandaceae family. This work will improve our understanding of the phylogeny of the Juglandaceae family. Materials And Methods Plant materials A total of 51 taxa were included in the analysis. A total of 21 accessions were collected in China, which included nineteen ingroup taxa of accessions collected from different locations, including senile Page 3/23 germplasm, wild germplasm, and Pterocarya hupehensis. The last was Juglans nigra (introduced from the USA). The ITS sequence of other genera was downloaded from the GeneBank of NCBI (http://www.ncbi.nlm.nih.gov ) (Table 1). Page 4/23 Table 1 Species list, collection site, geographic origin, and GenBank accession number of the 51 Juglandaceae species used for phylogenetic analysis Species list Collection Species/origin Description GenBank accession site number/ provider 21 Juglans regia Shannxi China old cultivars 9 Juglans regia Hutian Xinjiang old cultivars China 13 Juglans regia Wensu Xinjiang old cultivars China 16 Juglans regia Ili Xinjiang old cultivars China 28 Juglans regia Jining China old cultivars 14 Juglans regia Ruoqiang Xinjiang old cultivars China 23 Juglans regia Wenquan Gansu China cultivars 18 Juglans regia Ili Xinjiang wild walnut China 15 Juglans regia Kuche Xinjiang old cultivars China 11 Juglans regia Yecheng Xinjiang old cultivars China 17 Juglans regia Ili Xinjiang wild walnut China 10 Juglans regia Hutian Xinjiang old cultivars China 12 Juglans regia Yecheng Xinjiang old cultivars China 25 Juglans regia Jinin China old cultivars 19 Juglans regia Ili Xinjiang wild walnut China 27 Juglans regia Tibet China wild walnut * 8 Juglans regia EF141011 24 Juglans regia Wensu Xinjiang cultivars China * provided by the National Fruit Germplasm Tai’an Walnut & Chestnut Repositor. Page 5/23 Species list Collection Species/origin Description GenBank accession site number/ provider 26 Juglans Yunnan China landraces * sigiliata Juglans cinerea AF338480 22 Juglans Shannxi China Landraces mandshurica Juglans Hebei China landraces * hopeiensis 42Juglans AF179576 mandshurica Juglans major AF338480 Juglans neotropica AF179578 Juglans australis AF179568 Juglans AF179573 guatemalensis Juglans hindsii AF338477 Juglans californica AF338475 Juglans olanchana AF179580 Juglans AF338485 microcarpa 5Juglans nigra AF338490 20 Juglans nigra Ili Xinjiang Introduction China from USA Cyclocarya AF303817 paliurus Pterocarya Hubei China landraces hupehensis Pterocarya AF303814 macroptera Pterocarya AF303816 stenoptera Platycarya AF303808 strobilacea Carya glabra AF303823 * provided by the National Fruit Germplasm Tai’an Walnut & Chestnut